This invention relates to a method and system compensating for intensity fluctuations of an illumination system in a confocal microscope. More particularly, the invention relates to a method and system capable of carrying out compensation for intensity fluctuations of light in real time.
In confocal microscopy a specimen is scanned with a focused laser beam. The focus of the laser beam is moved in a section plane of a specimen by two scan mirrors tilting around their respective axes, which axes are perpendicular to each other. The first scan mirror diverts the laser light in the x-direction and the second scan mirror diverts the laser light in the y-direction while the intensity of the reflected or the fluorescent light is measured for each scanning point. Each measured intensity value relates to an x, y and z-position of the specimen, therefore, providing a user with a three-dimensional image of the specimen.
An article in the Journal of Microscopy, Vol. 117, Pt 2, November 1979, pp 233-242 by G. J. Brakenhoff, entitled xe2x80x9cImaging Modes in Confocal Scanning Light Microscopyxe2x80x9d, discloses a method for eliminating short term fluctuations of the illumination intensity by using a beam splitter to separate an illumination beam and a reference beam. In that method the ratio between the illumination beam and the reference beam is used to eliminate intensity fluctuations and create a better quality image.
An example for a confocal microscope is disclosed in U.S. Pat. No. 5,804,813 xe2x80x9cDifferential confocal microscopyxe2x80x9d to J. D. Wang et al. That Patent describes a Hexe2x80x94Ne laser as the light source, and a microscope objective lens as a focusing device. The light signal reflected from the surface of a specimen travels through a beam splitter and is almost completely reflected at the surface to travel to an optical detector which can use photodiodes, avalanche photodiodes, photo multipliers, charge coupled devices (CCDs), or fluorescent screens. The signal is detected by the optical detector and then amplified by a signal amplifier. The amplified signal is recorded by an analog-to-digital converter and then stored in a computer. The computer generates a three-dimensional image by using the intensity of the signal corresponding to the respective coordinates of the specimen. Before the measurement is performed, it is necessary to use the same sample to calibrate the relationship between the variation of signal intensity and the height of the sample.
Registration and processing of a position signal is normally done with analog circuits, computers or digital signal processors (DSP). There are certain disadvantages of performing signal processing with analog circuits. For example, signal processing can only be carried out with the help of a correction function implemented in an analog circuit. Compensation for intensity fluctuations is done in the analog circuit by the division of the signal from the specimen by the reference signal. If a modified or more complex correction function is to be used, a new design of an analog circuit may be necessary. Such more complex correction function can be, for example, the weighing of the signals to consider offsets or leveling the nonlinearity of the detectors. Changes of the correction mechanism, such as for example, changes of a scan rate, require a lot of effort. Moreover, the accuracy of analog circuits with respect to mathematical operations reaches its limit at high scan rates. Pixel rates greater than 1 MHz at a 12-bit accuracy can be achieved only with enormous effort.
It is an object of the present invention to provide a method for correcting intensity fluctuations of the illumination system of a confocal microscope in real time. Furthermore, it is also an object of the present invention to make the processing of the electric signals possible at high scanning rates. These objects are achieved by a method comprising the steps:
digitizing a first electric signal representing the light reflected from a specimen, and digitizing a second signal representing an illumination reference;
feeding the first and second electric signals to a first and second look up tables, respectively;
correcting the first electric signal for intensity fluctuations of the second electric signal; and
providing the corrected electric signal to a third look up table.
It is also an object of the present invention to provide a system capable of correcting intensity fluctuations of an illumination system in a confocal microscope. The system allows processing of electric signals at high scanning rates in real time. Additionally, it is an object of the present invention to allow a user to modify the existing processing algorithms easily.
These objects are accomplished with a system, comprising:
a first and a second analog-to-digital converters for digitizing a first electric signal corresponding to the light reflected from a specimen, and digitizing a second signal corresponding to an illumination reference, respectively;
a first and a second look up tables for converting the first and second electric signals, respectively;
a calculator for correcting the first electric signal for intensity fluctuations of the second electric signal to provide a corrected signal;
a third look up table for converting the corrected electric signal.
An advantage of the inventive method and system is that the position signal of the focused scanning beam and of the signals from the detectors (the scanning beam and reference beam) are digitized at a very early stage. Processing of the data is done mainly in a digital form with the use of a programmable control and processing unit which is implemented in a programmable digital circuit, such as, for example, FPGA (Field Programmable Gate Array). Correction parameters can be used online, making the subsequent image processing much easier or unnecessary at all. The accuracy of the device depends solely on the accuracy of the detectors and of the analog-to-digital converters. Analog-to-digital converters with a large processing bandwidth are available at reasonable costs. Modification of the scan device and scan elements (for example, by using new detectors) and also an increase in accuracy (for example, from 12 Bit to 16 Bit sampling) do not require changing or modifying the mechanical parts of the microscope. The present invention makes it possible to load new or upgrade existing filter modules or algorithms during data processing. The dynamic pixel accumulation during a measurement allows the system to operate in a wide dynamic range of scanning rates from a few Hz to up to 250 MHz.
A further advantage of the system is its ability to compensate intensity fluctuations in real time, which repeats the predetermined process steps with the accuracy up to nanoseconds. The system of the present invention satisfies the requirements of flexibility and real time data processing, because, for example, there is no need to buffer data, since a computer controlled data recording is not used. The real time processing ability of the inventive system provides a loss free and flexible data collection with the highest possible scan rates. Computers are controlled by interrupts, so a running process has to be ended before further data processing can go on. The present invention has the ability of a high flexibility with respect to data processing changes and real time processing of the data.